Synthesis and Identification of P22/P221 Polypeptides of IBDV Ligand Epitope and Screening CEF Library
|School||Henan Agricultural University|
|Course||Preventive Veterinary Medicine|
|Keywords||IBDV peptide synthesis phage library receptors|
IBD (Infectious Bursal Disease, IBD) is a highly infectious and transmitted disease for chickens. The bursa of Fabricius is the target organ of this virus. Classical IBDV strains could infect chickens aged 3 to 6 weeks and can induce immune deficiency, thus cause other pathogenic infections and high mortality, which lead to huge economic losses in poultry industry. IBDV viruses encode 5 kinds of proteins, and VP2 plays a crucial role as the major structural protein. Based on the current research of the VP2 protein of IBDV and its three-dimensional structure, we designed and synthesized a series of VP2 peptides. The series VP2 epitope peptides were screened and identified, which confirmed that ligand binding epitope of IBDV was P22. By deeply analysed P22 peptides, we got the results that P221 was the main affection part of P22 peptide, and had the same effect as peptides P22. P22 and P221 were synthesized in the research. They were used to study ligand epitope in DT40 cells and screening the receptor.For further studying the biological function of polypeptide and the application of receptors for IBDV, P22 and P221 were synthesized using Fmoc solid phase method in this experiment. The results were identified by mass spectrograph (MS) and biotin labeled peptides binding CEF experiment, it showed the peptides were obtained successfully.IBDV can infect B lymphocytes in chickens. Our study analyzed the synthetic peptides by using binding experiments and virus blocking binding assay on DT40 cells through Biotin—Avidin-HRP system in order to check the effects of peptides on DT40 cells. The results indicated P22 peptide and P221 peptide could effectively integrate with DT40 cells, and the more peptides, the stronger ability to bind DT40 cells. P22 and P221 could still integrate with DT40 cells by virus blocking experiment, but binding fraction was weak comparing with cell binding experiment, and the binding fraction decreased 70% (P22)and 50%(P221). The results confirmed that IBDV could effectively block the P22 and p221 to bind DT40 cells, which suggested P22 and P221 could bind B lymphocytes and was meaningful to understand the inter action between IBDV and its target cells.To study the mechanism of IBDV receptor and its target cell, we screened the cDNA library of CEF three times in the laboratory using mixed polypeptides of P22 and P221 and IBDV (XIN-1), the titers of phage were stable, which were 5.3×10~3pfu/mL、3.7×10~6pfu/mL、1.7×10~6 pfu/mL respectively. The input-output ratio were 1.9×10~7、2.7×10~4、5.8×10~4 respectively. The titers of phage after screening three times by P22 were 7.0×10~3pfu/mL、2.1×10~6pfu/mL、1.3×10~6pfu/mL respectively, The input-output ratio were 1.4×10~7、4.7×10~4、7.6×10~4respectively. The interaction between IBDV and the expression protein was proofed preliminarily by Phage-ELISA and Plaque lift. The useful plaques which could bind IBDV were selected and amplified by PCR. Blast software showed the selected sequence screened by IBDV was on ChEST898p20 of Gallus and the highest homology was 97%. Selected sequence screened by mixed polypeptides was on mRNA ofα2 chain of chicken collagen type I type, the highest homology was 97%. The protein expressed by such sequences might be the important binding site on CEF, which provided foundation to further study the receptor of IBDV.In this study, two ligand combining epitope named P22 and P221 were gained successfully and were verified that they could bind CEF effectively. Virus blocking experiment showed P22 and P221 could integrate with DT40 cells and the combination for polypeptides and DT40 cells could be blocked by IBDV. Further study indicated that some positive clones were obtained by screening the cDNA library of CEF using mixed polypeptides and IBDV. Two sequences were meaningful for the research and needed to study deeply.